Armor plate changes in sticklebacks are a classic example of repeated adaptive evolution. Previous studies identified ectodysplasin (EDA) gene as the major locus controlling recurrent plate loss in freshwater fish, though the causative DNA alterations were not known. Here we show that freshwater EDA alleles have cis-acting regulatory changes that reduce expression in developing plates and spines. An identical T ? G base pair change is found in EDA enhancers of divergent low-plated fish. Recreation of the T ? G change in a marine enhancer strongly reduces expression in posterior armor plates. Bead implantation and cell culture experiments show that Wnt signaling strongly activates the marine EDA enhancer, and the freshwater T ? G change reduces Wnt responsiveness. Thus parallel evolution of low-plated sticklebacks has occurred through a shared DNA regulatory change, which reduces the sensitivity of an EDA enhancer to Wnt signaling, and alters expression in developing armor plates while preserving expression in other tissues.